Li et al. Cell Death and Disease (2020) 11:509 https://doi.org/10.1038/s41419-020-2699-2 Cell Death & Disease ARTICLE Open Access SPEN induces miR-4652-3p to target HIPK2 in nasopharyngeal carcinoma Yang Li1,YuminLv1, Chao Cheng2,YanHuang3,LiuYang1, Jingjing He1,XingyuTao1, Yingying Hu1,YutingMa1, Yun Su1,LiyangWu1,GuifangYu4, Qingping Jiang5,ShuLiu6,XiongLiu7 and Zhen Liu1 Abstract SPEN family transcriptional repressor (SPEN), also known as the SMART/HDAC1-associated repressor protein (SHARP), has been reported to modulate the malignant phenotypes of breast cancer, colon cancer, and ovarian cancer. However, its role and the detail molecular basis in nasopharyngeal carcinoma (NPC) remain elusive. In this study, the SPEN mRNA and protein expression was found to be increased in NPC cells and tissues compared with nonmalignant nasopharyngeal epithelial cells and tissues. Elevated SPEN protein expression was found to promote the pathogenesis of NPC and lead to poor prognosis. Knockdown of SPEN expression resulted in inactivation ofPI3K/AKT and c-JUN signaling, thereby suppressing NPC migration and invasion. In addition, miR-4652-3p was found to be a downstream inducer of SPEN by targeting the homeodomain interacting protein kinase 2 (HIPK2) gene, a potential tumor suppressor that reduces the activation of epithelial–mesenchymal transition (EMT) signaling, thereby reducing its expression and leading to increased NPC migration, invasion, and metastasis. In addition, SPEN was found to induce miR-4652-3p expression by activating PI3K/AKT/c-JUN signaling to target HIPK2. Our data provided a new molecular mechanism for SPEN as a metastasis promoter through activation of PI3K/AKT signaling, thereby stimulating the c-JUN/miR-4652-3p axis to target HIPK2 in NPC. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction cause of treatment failure3. Accordingly, understanding Nasopharyngeal carcinoma (NPC) is highly prevalent in the molecular mechanisms by of invasion and/or metas- Southern China with a much higher incidence than tasis of NPC is critical for the identification of novel elsewhere1. It is characterized by high invasion and early therapeutic targets and formulation of better treatment metastasis. Patients with NPC are often diagnosed at strategies. advanced stage of the disease2. Although regional control In previous studies, several genes have been reported to – has been greatly improved by the advances in radio- be involved in NPC metastasis4 6. SPEN family tran- therapy and chemotherapy, metastasis remains the major scriptional repressor (SPEN), also known as SMART/ HDAC1-associatedrepressor (SHARP), is a large nuclear protein that plays an important role in transcriptional Correspondence: Xiong Liu ([email protected])or regulation and inactivation of chromosome X7. Legare Zhen Liu ([email protected]) et al. reported that the inactivation of SPEN may con- 1Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein tribute to breast tumor progression and thus suggested Modification and Degradation, State Key Laboratory of Respiratory Disease, SPEN as a tumor suppressor in ERα-positive breast can- School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou cers8. In contrast, Feng et al. found that SPEN (SHARP) 510095 Guangdong, China 2Department of Pediatric Otorhinolaryngology, Shenzhen Key Laboratory of gene acts as a candidate oncogene, promoting the Viral Oncology, The Clinical Innovation & Research Centre, Shenzhen Hospital, pathogenesis of human hematopoietic malignancies, Southern Medical University, Shenzhen, Guangdong, China breast and colon cancer9. Furthermore, Liu et al. Full list of author information is available at the end of the article These authors contributed equally: Yang Li, Yumin Lv, Chao Cheng, Yan Huang demonstrated that SPOCD1(SPEN) may act as a Edited by G. Calin © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Li et al. Cell Death and Disease (2020) 11:509 Page 2 of 12 carcinogenesis factor by activating the PI3K/AKT path- SPEN through the activation of PI3K/AKT/c-JUN sig- way to restrained cell apoptosis in Ovarian cancer (OC)10. naling. In addition, miR-4652-3p was found to directly These studies suggested that SPEN played a significant target HIPK2 to participate in the SPEN-mediated pro- and complexed role in tumor pathogenesis. However, the motion of NPC migration, invasion, and metastasis. molecular alterations and biological functional involve- ment of SPEN in the pathogenesis of NPC have not been Results investigated. SPEN expression and clinicopathological characteristics in MicroRNAs (miRNAs) area class of small (17–23 NPC nucleotides) noncoding RNAs that silence mRNA mole- To determine the role of SPEN in NPC development, its cules through a degradation or translational inhibition expression level was analyzed in various NPC cell lines process. They participate in various biological processes, (HONE1, SUNE1, 5-8F, 6-10B, CNE1, and CNE2) and – including tumorigenesis and metastasis11 13. Multiple immortalized nasopharyngeal epithelial (NP) cell lines miRNAs have been found to play key roles in regulating (NP69 and SXSW-1489) by quantitative real-time poly- the expression of various critical genes during the devel- merase chain reaction (qRT-PCR) analysis. The endo- opment of human tumors4,14,15. Several of them were genous mRNA level of SPEN in all six NPC cell lines was identified as regulators of the progression of NPC, such as significantly upregulated compared with that in SXSW- miR-374a, miR-184, and miR-31886,16,17. However, the 1489 nonmalignant immortalized NP cells, although the regulation of miRNAs involving SPEN has not been difference between NPC cells and NP69 nonmalignant NP reported to date. cells (Fig. 1a) was not significant. As for protein level, a This study reports a newly discovered miRNA, namely large cohort of 238 NPC tissues and 54 nonmalignant miR-4652-3p, as an oncogenic regulator miRNA, which NP tissues were examined by immunohistochemistry was found to be upregulated by the potential oncogene (IHC) analysis. SPEN expression displayed nuclear and Fig. 1 Elevated expression of SPEN promoted the poor prognosis of NPC patients. a mRNA levels of SPEN analyzed by qRT-PCR assays in six human NPC cell lines (HONE1, SUNE1, 5–8F, 6-10B, CNE1, CNE2) and immortalized normal nasopharyngeal epithelial cell lines NP69 and SXSW-1489. b Representative IHC images of SPEN expression in NP and NPC tissues. a, b: weak expression of SPEN in NP samples; c, d: strong and positive expression of SPEN in NP samples; e, f: weak staining of SPEN in NPC specimens; g, h: strong and positive staining of SPEN in NPC specimens. (original magnification ×400). c Kaplan–Meier survival curve for overall survival in NPC patients based on SPEN expression level (P = 0.0305, log-rank test). Official journal of the Cell Death Differentiation Association Li et al. Cell Death and Disease (2020) 11:509 Page 3 of 12 Table 1 The expression of SPEN in NPC compared with SPEN induces miR-4652-3p expression in NPC cells NP tissues. To investigate the downstream effector miRNAs regu- lated by SPEN, an Affymetrix 3.0 miRNA array was used Group Cases (n) SPEN expression χ2 P* value to examine the differential expression of miRNAs between HONE1-siSPEN and HONE1-NC cells (Fig. 3a). High Low Expression analysis by qRT-PCR confirmed that miR- expression expression 4652-3p expression was downregulated by twofold or NPC 238 141 (59.2%) 97 (40.8%) 29.234 0.000 more at the mRNA level in SPEN-silenced HONE1 cells compared with HONE1-NC group (Fig. 3b). NP 54 10 (18.5%) 44 (81.5%) NPC nasopharyngeal carcinoma, NP nasopharyngeal epithelium. MiR-4652-3p promotes NPC cell metastasis in vitro and *χ2 test was applied to assess the expression of SPEN in NPC and NP in vivo HONE1 and 5–8F cells successfully transfected with cytoplasmic distribution patterns in both NPC and NP miR-4652-3pmimics were used to investigate the effects cells with different expression levels (Fig. 1b). Statistical of miR-465-3p on migration and invasion alterations analysis confirmed that among the 238 NPC specimens, in vitro. First, qRT-PCR analysis showed the expression 97 (40.8%) had low SPEN expression and 141 (59.2%) had level of miR-4652-3p was higher in HONE1 and 5–8F high SPEN expression. Instead, among the 54 NP tissues, cells transfected with miR-4652–3p mimics than that in low SPEN-expressing tissues accounted for 44 (81.5%), cells transfected with NC (Fig. 3c). In addition, over- and high SPEN-expressing tissues accounted for 10 expression of miR-4652-3p in HONE1 and 5–8F cells (18.5%). In addition, NPC tissues showed higher SPEN greatly promoted cell migration and invasion abilities (Fig. expression level than NP tissues(P < 0.001, Table 1). In 3d, e). In addition, we established two NPC cell lines addition, the analysis of the relationship between SPEN stably overexpressing miR-4652–3p, namely HONE1-LV- expression and the clinicopathological characteristics in miR-4652-3p and 5-8F-LV-miR-4652-3p, by lentivirus- patients with NPC revealed no statistically significant mediated transfection and used them for in vivo studies.